Scripts
The code used for analysis is located on GitHub and compiled
together here.
Quality
control
Calling nuclei
barcodes
- First, we used Cell Ranger ARC called cell barcodes – algorithm
described here.
14,301 barcodes out of 2,215,183.
- Second, because the Cell Ranger ARC cell calling algorithm is very
permissive to barcodes with very low counts (i.e., a minimum of
a single count in each library), barcodes were additionally filtered to
a low count threshold in both the ATAC and RNA libraries based on the
clearly defined population of cells in the RNA and ATAC count
scatterplot. Additionally, barcodes with more than 5% of RNA reads
mapping to genes on the mitochondrial genome were excluded. A total of
886 barcodes were filtered out for 13,145 left.
- Last, multiplets were filtered out using two independent methods,
relying on either the ATAC or RNA libraries to call multiplets. AMULET relies on the
assumption that in snATAC-seq of diploids there should be at most two
overlapping fragments with the same cell barcode. The presence of more
than two overlapping fragments is a potential indication of a multiplet.
Doublets were also identified using the RNA-seq libraries with DoubletFinder.
An additional 1,103 multiplet barcodes filtered out, for a final number
of 12,042 high quality nuceli barcodes.
Knee plot

Scatter plot of counts
Cells after all filtering

Cell Ranger ARC only

And low count threshold

Violin
QC plots
After filtering

Before filtering

Multiplets
Mapped onto UMAP projection

Counts

Count histograms
Multiplets

After filtering

Seurat
Clusters
Cells per cluster

Acclimation clusters
Together

Split
